This project is a study of the mechanism of action of cyclic AMP as a mediator of catecholamine effects at beta-adrenergic receptors. Protein phosphorylation will be studied in intact isolated heart cells (myocytes) and in nucleated erythrocytes by incubation with 32Pi, treatment with beta-adrenergic agonists and related drugs, and analysis of cellular phosphoproteins by polyacrylamide gel electrophoresis. Parallel studies will be conducted on 45 Ca ions influx with myocytes in order to correlate changes in protein phosphorylation with changes in cellular physiology. Similarly, studies on sodium and potassium fluxes will be carried out with nucleated erythrocytes, since this is the physiological response of these cells to beta-adrenergic stimulation. Also, purification of a catecholamine-sensitive phospho-protein from avian erythrocyte membranes will be attempted, to provide further information about its relationship to cyclic AMP-dependent ion fluxes. Experiments will also include identification and subcellular localization of cyclic AMP receptors by covalent photoaffinity labelling with (32P)-8-axidocyclic AMP. It is anticipated that this project will increase our knowledge of how hormones act at the molecular level to regulate membrane in the heart and other tissues.